RT-PCR is one of the easiest ways to confirm gene profiling and gene function analyses generated by methods such as array analysis and siRNA experiments, especially when there are many samples to analyze. However, there is one catch. For RT-PCR experiments, total RNA must be isolated first. Why not skip this step? Ambion scientists now demonstrate the use of the Cells-to-cDNA™ II Kit to produce real time RT-PCR data quickly, with minimal effort. Cells-to-cDNA cell lysates can be used for RT-PCR directly without RNA isolation. In this article, we show how the Cells-to-cDNA™ II Kit can be used to assess siRNA experiments for the development of an optimized RNA interference protocol. In addition to analyzing siRNA data, Cells-to-cDNA can be used to process FACS and LCM samples. The kit has also been adapted to 96 well plate automated processing. See "Automated Cells-to-cDNA Protocol" for the procedure.

AM1620,AM1722,AM1723,AM4502,AM4503

Experimental Set-up: siRNA Transfection

HeLa cells were transfected with siRNA and an automated protocol for the Cells-to-cDNA II Kit was used to look at specificity of the siRNA effect and optimal cell concentration.

HeLa cells were examined under three experimental conditions: 1) cells that were transfected with a GAPDH siRNA, 2) cells that were transfected with a negative control siRNA and 3) untransfected cells. To optimize cell plating density for transfection of HeLa cells, a 96 well plate format was utilized. The plate was divided into 3 sections. Each section corresponded to one of the three experimental conditions outlined above. Each individual row contained the same number of initial cells. The cells were diluted 2 fold from the top of the plate to the bottom starting with 64,000 cells and ending with 1000 cells. This gave 4 replicates for each experimental condition.

siRNAs were made using the
Silencer™ siRNA Construction Kit. Both GAPDH siRNA and negative control siRNA (included in the Silencer siRNA Construction Kit) were transfected into cells at a final concentration of 10 nM using siPORT™Lipid Transfection Agent. Fresh medium was added 4 hours after transfection.

The cell culture plate was transferred to the deck of a Perkin Elmer MultiPROBE® II robotic platform 48 hours post transfection. The automated Cells-to-cDNA II protocol was performed followed by one-step real-time RT-PCR using a TaqMan® probe and primers for GAPDH. The robot removed the growth media, washed the cells with PBS, and then added 100 µl of Cell Lysis Buffer. The plate was moved to a heating tile, which brought the samples to 75°C for 12 min, simultaneously disrupting the cells and inactivating RNases. The samples were treated with DNase I followed by heat inactivation. Lysate (5 µl) was transferred robotically to a 96 well PCR plate containing 20 µl of a one-step real-time RT-PCR master mix.

This analysis determined that the optimal cell concentration necessary to induce maximum interference was 2000 cells per well (Figure 1). Data clearly show that the optimal number of HeLa cells plated in this experiment was 2,000 cells (Figure 1). At this plating density, cells transfected with the siRNA targeting GAPDH expressed only 30% the GAPDH levels of those transfected with the negative control siRNA.

For more information about the Cells-to-cDNA II automated protocol, see "Automation of One-Step RT-PCR Using Ambion's Cells-to-cDNA™ II Kit and the MultiPROBE® II HT Liquid Handling System".